Spirohydantoin mustard (SHM) has shown curative activity against the murine intracerebral ependymoblastoma and is currently undergoing preclinical toxicology studies prior to clinical trials. We have discovered that SHM, which has little or no mutagenic activity in vitro, is activated in vivo by liver microsomal oxygenases to a mutagen that is also, like SHM, an alkylating agent. This has led us to propose that this metabolite, tentatively identified as spirohydantoin aziridine (SHAZ), may be a dominant mediator of SHM anticancer activity in vivo. Consequently, the objective of the proposed research is to investigate the role of SHAZ in the mechanism of action of SHM. The following studies will be done using established methodologies: (1) chemical synthesis of (14C)-labeled SHM and SHAZ, (2) confirmation of the chemical structure of SHAZ (TLC; mass spectrometry) by comparison of synthetic SHAZ with isolated metabolite, (3) stability of SHAZ in buffer, plasma, and urine (TLC and radioassay), (4) determination of the LD50 and LD90 of SHAZ and SHM against murine leukemia P388 cells in culture, (5) a study of the disposition and metabolic profiles (plasma, urine, brain) of SHM and SHAZ in mice after iv administration of each drug at two dose levels (TLC and radioassay), (6) a study of the chemotherapeutic activity of SHAZ against intraperitoneal and intracerebral P388 leukemias, intracerebral ependymoblastoma, Lewis lung tumor, one murine mammary tumor, and one murine colon tumor. These studies could provide for the identification of an anticancer agent that is superior to SHM.